Overactive RAS signaling can drive cellular metabolic insufficiencies that promote a macropinocytic phenotype. In glioblastoma cells, uncontrolled oncogene-driven macropinocytosis can lead to methuosis, an understudied non-apoptotic form of cell death involving cell rupture due to an inability to process internalized macropinosomes. While this phenotype had previously only been shown to occur in glioblastoma cells in response to ectopic expression of constitutively active RAS, we recently demonstrated that EGFR amplification can also drive methuosis. This important observation may hold clues for new methods to treat glioblastoma because EGFR is frequently over-expressed in tumors. We hypothesize that some glioblastoma cells may be able to adapt to EGFR-driven methuosis stress and that learning how cells cope with the stress and process macropinosomes may reveal new therapeutic vulnerabilities for the treatment of glioblastoma. We propose that studying cytoskeletal dynamics and structure is an ideal way to pursue that hypothesis and deepen our understanding of the connection between EGFR and methuosis in glioblastoma. Indeed, the cytoskeleton is intimately involved in the formation and trafficking of macropinosomes, and both EGFR and RAS actively control the cytoskeleton at multiple levels. Our preliminary observations suggest that disrupting the intermediate filament and tubulin networks with inhibitors can forestall methuosis and that macropinosome accumulation leads to clear displacements of the normal cytoskeleton within cells. However, it is yet unclear if cytoskeletal perturbations (at all three levels of intermediate filaments, actin, and tubulin) precede and promote methuosis or occur as a consequence of methuosis. The Lazzara lab recently showed that over-expression of EGFR, a common occurrence in glioblastoma, promotes methuosis, an understudied form of cell death resulting from uncontrolled macropinocytosis leading to cell rupture. The cytoskeleton is intimately involved in macropinosome formation and trafficking, but the role of the cytoskeleton in methuosis has never been studied. In this project, a cancer systems biologist with expertise in EGFR signaling will collaborate with a cell biologist with expertise in live-cell imaging to determine if perturbations to the glioblastoma cell actin, tubulin, and intermediate filament networks lead to or result from methuosis, which may reveal methods to promote the phenotype therapeutically.